Galvinoxyl radicals: Synthesis of new derivatives, determination of low oxygen contents, and stability studies

Article abstract of DOI:10.1016/j.tet.2019.03.051

Two new derivatives of galvinoxyl (1), a perdeutered (2) and an adamantyl-analog (3) for potential applications as spin probes were synthesized. The synthesis with deuterated educts yielded 2 with 98% D. It exhibited an 18-line EPR spectrum in octanol wit

Full text of DOI:10.1016/j.tet.2019.03.051

Tetrahedron 75 (2019) 2737e2747
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Galvinoxyl radicals: Synthesis of new derivatives, determination of
low oxygen contents, and stability studies
a
b
b
a, *
Lisa Lampp , Mykhailo Azarkh , Malte Drescher , Peter Imming
a
Institute of Pharmacy, Martin Luther University Halle Wittenberg, Wolfgang-Langenbeck-Str. 4, 06120 Halle Saale, Germany
Department of Chemistry and Konstanz Research School Chemical Biology, University of Konstanz, Universit a€ tsstraße 10, 78464 Konstanz, Germany
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 28 February 2019
Received in revised form
Two new derivatives of galvinoxyl (1), a perdeutered (2) and an adamantyl-analog (3) for potential
applications as spin probes were synthesized. The synthesis with deuterated educts yielded 2 with 98%
D. It exhibited an 18-line EPR spectrum in octanol with narrow peak-to-peak linewidth. The EPR spec-
trum of 3 was very similar to galvinoxyl, but with differences in the linewidth due to unresolved long-
23 March 2019
Accepted 26 March 2019
Available online 30 March 2019
range couplings with adamantyl-protons. Compound 2 showed a higher response to oxygen (4.8
2
mT/% O )
than 1 (2.8 T/% O ). The coupling pattern of 2 allowed the determination of oxygen at low levels (0e6%)
m
2
by a new type of analysis of the EPR pattern. The stability of the radicals strongly depended on the
amount of hydrogalvinoxyl, a by-product of the galvinoxyl synthesis, and the solvent type. A molecular
mechanism for the stabilization by hydrogalvinoxyl and the influence of solvent type is proposed.
© 2019 Elsevier Ltd. All rights reserved.
Keywords:
Galvinoxyl radicals
EPR
Oximetry
Mechanism of stabilization
1. Introduction
difficult to remove due to the very similar chromatographic
behavior [14]. The mechanism of stabilization is not known.
Galvinoxyl or “Coppinger's” radical (1, Fig. 1) was first synthe-
sized by Galvin M. Coppinger 1957 [1]. Since then the synthesis and
characteristics of many derivatives of galvinoxyl radical have been
published [2e4]. However, only a few derivatives are known with
other substituents then tert. butyl in the ortho position. Only ana-
logs with methyl or phenyl groups, but none with other sterically
hindered alkyl groups were published [5,6]. While partially
deuterated analogs of galvinoxyl radical were prepared [7e9], the
fully deuterated galvinoxyl radical has not been reported.
The aim of this study was to prepare a fully deuterated galvi-
noxyl radical and a galvinoxyl derivative with adamantyl groups
instead of tert.-butyl groups and examine their properties (com-
pound 2 and 3, Fig. 1). We further studied the oxygen sensitivity of
1 and 2 to prove their suitability as spin probes for EPR oximetry. In
view of applications of the radicals, we gained some insight into of
stabilization of galvinoxyl by hydrogalvinoxyl and examined which
other aspects influence the stability of galvinoxyl.
Little is known about the stability of galvinoxyl. In the presence
of reducing agents like ascorbic acid, glutathione or cysteine, the
radical is reduced immediately or within minutes [10]. Galvinoxyl
reacts with other short-lived radicals which makes it a useful
radical scavenger [11e13]. Coppinger stated that galvinoxyl is stable
towards oxygen not only as solid, but also in solution [1]. Later
authors found that pure galvinoxyl reacts quite fastly with oxygen,
yielding 2,6-di-tert-butyl-1,4-benzoquinone and 3,5-di-tert-butyl-
2
2
2
. Materials and Methods
.1. Synthesis
.1.1. Materials and general methods for synthesis
All chemicals used for synthesis were purchased and used
without further purification. All organic solvents were purified
before use. Column chromatography was performed on silica gel 60
4
-hydroxybenzaldehyde [14]. Stabilization of the radical against
(70e230 mesh or 230e400 mesh). The purity of all compounds and
oxygen can be achieved by hydrogalvinoxyl (1H, Fig. 11), an im-
purity that is formed during the synthesis of galvinoxyl and very
the progress of the reactions were monitored by thin layer chro-
matography using silica gel 60 F254 plates (Merck KGaA, Darm-
stadt, Germany). Visualizations were accomplished with an UV
lamp (254 nm) or iodine staining. The R
f
values given are uncor-
rected. H and C NMR spectra were recorded on an Agilent
Technologies VNMRS 400 MHz or Varian Inova 500 MHz
1
13
*
Corresponding author.
E-mail address: peter.imming@pharmazie.uni-halle.de (P. Imming).
a
https://doi.org/10.1016/j.tet.2019.03.051
040-4020/© 2019 Elsevier Ltd. All rights reserved.
0

2738
L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
Fig. 1. Structure of galvinoxyl (1), hydrogalvinoxyl (1H), perdeuterated galvinoxyl (2) and adamantyl-galvinoxyl (3).
spectrometer. Chemical shifts (
sidual solvent peak of CDCl as internal standard:
¼ 77.0 ppm. C NMR spectra of the deuterated compounds show
d
) are reported relative to the re-
124.68, 124.49, 124.30, 119.39, 119.11, 118.92, 33.65, 33.57, 30.30,
29.66, 29.50, 29.35, 29.20, 29.05, 28.90.
3
d
H
¼ 7.26 ppm,
13
d
C
more signals than expected. This is a result of coupling between
carbon and deuterium atoms and arises also from incomplete ex-
change of protons which leads to different deuteration patterns.
Infrared (IR) spectra were recorded on a Bruker IFS 28 FTIR spec-
trometer equipped with a Thermo Spectra-Tech attenuated total
reflection (ATR) unit with a 20 mm ZnSe-Fresnel crystal. High res-
olution mass spectra (HRMS) were measured on a LTQ-Orbitrap-XL
2
.1.3. Synthesis of 2 from deuterated educts
.1.3.1. 4-Bromo-2,6-bis[2-(D )methyl(1,1,1,3,3,3-D
)phenol (11). Compound 10 (1 g, 5.64 mmol) was mixed in a
glass pressure vessel with tert. butanol-d10 (2.66 ml, 28.24 mmol)
2
3
6
)propan-2-
yl](D
2
and D
2
SO
4
(150
m
l, 2.66 mmol). Vessel was tightly closed and
ꢀ
heated at 160 C for 3 h. After cooling to RT mixture was dissolved
in 50 ml CHCl
organic layer was dried over MgSO
3
and washed successively with water and brine. The
and solvent was evaporated to
(
ESI source) of Thermo Scientific. Samples were dissolved in
4
chloroform-methanol mixtures. Deuterium incorporation was
determined by NMR using hexamethylbenzene as internal standard
or by high resolution mass spectrometry.
dryness. The residue was purified with silica gel, eluting with
heptane/ethyl acetate (10/0 to 9/1). Compound 11 was isolated, all
other fractions containing unreacted or monosubstituted 10 were
evaporated and reaction was done again. The reaction was done
four times in total to give 500 mg (29% yield) 11 as a white to pale
2.1.2. Synthesis of 2 by H/D exchange
ꢀ
1
The substrate and catalysts (Method A: 5% Pd/C (20 wt%), 5% Pt/
yellow solid: mp 74e76 C, R ¼ 0.43 (heptane), H NMR (400 MHz,
f
13
C (20 wt%); Method B: 1% Pd/C (100 wt%), Pt/C (100 wt%)) in D
2
O
3 3
CDCl ): d 7.24 (s), 5.14 (s), 1.37 (s), C NMR (101 MHz, CDCl ):
ꢀ
were stirred at 180 C in an autoclave under H
2
atmosphere (1 atm)
d 152.89, 138.06, 129.46e125.73 (m), 112.36, 34.75, 30.09e27.86
-
for different time periods. The reaction mixture was diluted with
DCM or CHCl and then filtered to remove the catalysts. The residue
was washed several times with DCM or CHCl . The combined
organic layers were washed with water, dried over MgSO and were
(m), HRMS (ESI): calcd. for C14D20BrO [M ꢁ H] 303.196; found
3
303.196, degree of deuteration 97.7% (ESI-MS).
3
4
2
.1.3.2. Bis[2-(D
Compound 11 (456 mg, 1.49 mmol) dissolved in DCM/methanol
55 ml, 2/1) and 5% Pd/C (230 mg, 50 wt% of 11) were stirred under
atmosphere (8 bar) at RT for 7 h. Mixture was then filtered to
3 6 3
)methyl(1,1,1,3,3,3-D )propan-2-yl](D )phenol (9).
evaporated to dryness. The residue was purified by silica gel col-
umn chromatography.
(
D
2
2
.1.2.1. 4-(6,3,3,3-D
6
)Propan-2-yl](2,6-D
2
)phenyl(D
)phenol
2
)methyl)-2,6-bis
remove the catalyst. The filtered catalyst was washed several times
with DCM. The combined organic layers were evaporated to dry-
ness. The residue was purified by column chromatography on silica
gel, eluting with heptane/ethyl acetate (10/0.2) to give 340 mg (88%
[2-(D )methyl(1,1,1,3,3,3-D
3
6
)propan-2-yl](D
2
(6).
Method A: Compound 6 was not obtained. Method B (3 days): After
purification on silica gel eluting with heptane/CHCl (8/2) com-
pound 6 could be isolated as a pale yellow solid (18%). R
¼ 0.40
7.03 (d), 5.03 (d),
.88e3.77 (m), 1.47e1.32 (m), C NMR (126 MHz,CDCl ): 151.88,
51.86, 135.68, 135.66, 131.92, 125.32, 125.28, 41.03, 40.91, 40.83,
3
f
yield) of a colorless liquid: R ¼ 0.42 (heptane/ethyl acetate 10/0.2),
f
1
1
(
heptane/CHCl
3
8/2), H NMR (400 MHz, CDCl
3
):
d
3
H NMR (400 MHz, CDCl ): d 7.17 (s), 6.83 (s), 5.17 (s), 1.45e1.38 (m),
C NMR (101 MHz, CDCl ): d 153.84, 135.80, 124.72, 124.48, 124.24,
3
13
13
3
1
4
2
3
d
119.38, 119.10, 118.86, 33.55, 29.38, 29.19, 29.00, HRMS (ESI): calcd.
-
0.70, 34.31, 34.23, 30.33, 30.33, 29.84, 29.68, 29.54, 29.36, 29.25,
9.07. Degree of deuteration (NMR): 53% (for C29 , tert.
for C14D21O [M ꢁ H] 226.292; found 226.296, degree of deuteration
2
H D
42
O
2
97.7% (ESI-MS).
butyl 56%, ortho 5%, methylene bridge 95%). Method B (5 days):
After purification on silica gel eluting with heptane/CHCl (8/2)
compound 6 could be isolated as a pale yellow solid (4%). R
¼ 0.40
7.02 (s), 5.02 (s),
.90e3.78 (m), 1.44e1.21 (m), C NMR (126 MHz, CDCl ): 151.87,
35.65, 135.59, 131.92, 131.84, 131.75, 125.28, 125.17, 124.98, 124.81,
3
2
.1.3.3. 4-({4-Hydroxy-3,5-bis[2-(D
yl](2,6-D )phenyl}(D )methyl)-2,6-bis[2-(D
propan-2-yl](D )phenol (6). Compound 9 (197 mg, 0.867 mmol)
and paraformaldehyde-d (111 mg, 3.464 mmol) were dissolved in
ml isopropanol-d and 250 l D O under argon atmosphere. Then
the mixture was heated to 35 C and 100 l 40% NaOD in D O were
)methyl(1,1,1,3,3,3-D
6
)propan-2-
3
f
)methyl(1,1,1,3,3,3-D
6
)
2
2
3
1
(
3
1
3 3
heptane/CHCl 8/2), H NMR (400 MHz, CDCl ): d
2
13
3
d
2
2
1
m
ꢀ
2
4
2
C
0.85, 40.58, 40.37, 33.67, 33.59, 30.32, 29.85, 29.69, 29.53, 29.38,
9.23, 29.08, 28.96. Degree of deuteration (NMR): 87% (for
m
2
added in one portion. The mixture was stirred for 40 min. During
this time the color of the reaction mixture changed from colorless
H
29 2
D
42
O
2
, tert. butyl 90%, ortho 53%, methylene bridge 96%).
2 4
to dark red. Reaction mixture was then acidified with D SO until
2
.1.2.2. Bis[2-(D )methyl(1,1,1,3,3,3-D )propan-2-yl](D )phenol (9).
3
6
3
the color changed to yellow. Afterwards the mixture was diluted
with DCM. The organic layer was separated and the aq. layer was
washed several times with DCM. The combined organic layers were
Method A: Compound 9 was not obtained. Method B: After puri-
fication on silica gel eluting with pentane/DCM (10/0 to 8/2) 9 could
be isolated as a colorless liquid. Yield: 28% after 2 d, 18% after 3 d.
Degree of deuteration (NMR): 93% D (tert. butyl 95% D, ortho 69% D,
4
dried over MgSO and evaporated to dryness. The residue was
purified by column chromatography on silica gel eluting with
para 96% D) independent of reaction time. R
f
¼ 0.38 (heptane/CHCl
7.18 (s), 5.18 (d), 1.49e1.38 (m),
153.85, 135.88, 135.81, 124.87, 124.76,
3
heptane/CHCl
151e152 C, R
3
(8/2) to give 115 mg (57% yield) of white solid: mp
1
ꢀ
1
9
/1), H NMR (500 MHz, CDCl3):
d
f
¼ 0.39 (heptane/CHCl
3
8/2), H NMR (500 MHz,
1
3
13
C NMR (126 MHz, CDCl
3
):
d
CDCl3):
d
7.03 (s), 5.03 (s), 3.76 (s), 1.38 (s),
C NMR

L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
2739
(
4
2
126 MHz,CDCl
3
):
0.56, 40.43, 40.33, 40.27, 33.66, 33.59, 29.84, 29.68, 29.54, 29.38,
9.23, 29.07, 28.94, 28.78, HRMS (ESI): calcd. for C29
d
151.87, 135.59, 131.75, 125.17, 124.99, 124.80,
1.79 (s, 24H), 13C NMR (126 MHz, CDCl
3
):
d
152.55, 135.63, 132.05,
125.26, 41.47, 41.34, 37.06, 36.78, 29.09, HRMS (ESI): calcd. for
H
1
D
42
O
2
C
53
67
H O
2
[M ꢁ H]þ 735.514; found 735.515.
-
[
M ꢁ H] 465.591; found 465.599, degree of deuteration 96.0% (ESI-
MS).
2
.1.4.4. [2,6-Bis(adamantan-1-yl)-4-{[3,5-bis(adamantan-1-yl)-4-
methyl}phenyl]oxidanyl (3).
(129 mg, 0.391 mmol) was dissolved in 5 ml water. 1 ml
0% NaOH in water and 15 ml benzene were added. Mixture was set
oxocyclohexa-2,5-dien-1-ylidene]
Fe(CN)
2
.1.3.4. [4-({3,5-Bis[2-(D
3
)methyl(1,1,1,3,3,3-D
)cyclohexa-2,5-dien-1-ylidene}(D)methyl)-2,6-bis[2-(D
)propan-2-yl](3,5-D )phenyl]oxidanyl (2).
(246 mg, 0.748 mmol) were dissolved under argon in
O. 1.3 ml 40% NaOD in D O and 6 ml benzene-d were
added. Compound 6 (97 mg, 0.208 mmol) was dissolved in 15 ml
benzene-d and was added dropwise to the reaction mixture. The
6
)propan-2-yl]-4-
K
4
3
6
oxo(2,6-D
2
3
)
methyl(1,1,1,3,3,3-D
Fe(CN)
.5 ml D
6
2
under argon atmosphere. Compound 17 (80 mg, 0.109 mmol) was
dissolved in 60 ml benzene and was added dropwise to the reaction
mixture. After completely addition of 17 the mixture was stirred for
another 60 min during which the color of the mixture turned dark
red to brown. The organic layer was then separated, washed with
K
3
6
6
2
2
6
6
reaction mixture was stirred for another 60 min at RT under argon
atmosphere. The organic layer was then separated and washed
with water until the aq. phase was colorless. The organic layer was
dried over MgSO and was evaporated to dryness. The dark blue
4
product (97 mg, 100% yield) was used without further purification.
4
water until the aq. layer was colorless, dried over MgSO and was
evaporated to give 100 mg (125% yield) of dark violet solid. NMR
analysis showed no residual educt or other sideproducts, but a
benzene signal (7.36 ppm) which explains the surplus of 25%. The
residual benzene could not be removed under vacuum. 3 was used
ꢀ
Mp 150e153 C, R
f
¼ 0.18 (heptane/ethyl acetate 10/0.2), HRMS
þ
without further purification for the following experiments.
(
ESI): calcd. for C29HD41
O
2
[MþH] 463.576; found 463.575; degree
ꢀ
Mp ꢂ 250 C (degradation), R
f
¼ 0.31 (heptane/CHCl
3
8/2), HRMS
[M] 733.499; found 733.497, IR (ATR):
004, 2966, 2898, 2845, 2675, 2655, 1573, 1510, 1453, 1341, 1312,
of deuteration 98% (ESI-MS), IR (ATR): 3619, 3550e3208, 3114,
929, 2205, 2132, 2088, 2069, 2047, 1655, 1606, 1564, 1529, 1490,
409, 1319, 1280, 1254, 1209, 1159, 1131, 1061, 1047, 1008, 958, 905,
þ
(
65 2
ESI): calcd. for C53H O
2
1
7
3
ꢁ1
ꢁ
1
1253, 1224, 1216, 1204, 1164, 1104, 1024, 983, 908, 827, 751 cm .
80, 742 cm
.
2
2
.1.4. Synthesis of 3
.1.4.1. 2,6-Bis(adamantan-1-yl)-4-bromophenol
2
2
4
.2. Characterization
(15).
Compound 12 (1.5 g, 8.67 mmol) was mixed with 1-adamantanol
.2.1. EPR spectroscopy
Measurements at defined oxygen contents were conducted in
ml glass vials using an EPR spectrometer at 1.3 GHz (Magnettech,
(
2.64 g, 17.34 mmol)
and
1-bromoadamantane
(392 mg,
ꢀ
1.82 mmol) in a glass pressure vessel and for 3 h at 210 C. After
cooling to RT the reaction mixture was purified by column chro-
matography on silica gel, eluting with heptane to give 1.18 g (30%
Berlin, Germany) equipped with a re-entrant resonator. Measure-
ments were done under ambient conditions without temperature
control. General settings were as follows: microwave power
ꢀ
1
yield) of a white solid: mp > 260 C, R
400 MHz, CDCl ):
2H), 13C NMR (126 MHz, CDCl
f
¼ 0.35 (heptane), H NMR
(
1
3
d 7.19 (s, 2H), 5.32 (s, 1H), 2.10 (s, 18H), 1.78 (s,
0
.42 mW; modulation frequency, 100 kHz; sweep, 0.75e2 mT
depending on linewidth); scan time, 600e1200 s. The modulation
amplitude was set so that no line distortions occurred. Stability
measurements were done in 50 l capillaries using an X-band EPR
3
):
d
153.58, 138.10, 127.75, 113.26,
(
þ
41.01, 36.94, 36.86, 28.93, HRMS (ESI): calcd. for C26
H
33BrO [M]
4
40.171; found 440.170.
m
spectrometer at 9.30e9.55 GHz (Miniscope MS 200, Magnettech,
Berlin, Germany). Measurements were done under ambient con-
ditions without temperature control. General settings were as fol-
lows: microwave power 3.162 mW; modulation frequency,
2
0
.1.4.2. 2,6-Bis(adamantan-1-yl)phenol
.453 mmol) was dissolved in 80 ml DCM/methanol (1/1). 5% Pd/C
atmo-
(16). 15
(200 mg,
(
100 mg, 50 wt%) were added. Mixture was set under H
2
sphere (2 bar) and was stirred overnight at RT. The reaction mixture
was then filtered to remove the catalyst. The residue was washed
with DCM. Combined organic layers were evaporated to dryness.
The reaction mixture was purified by column chromatography on
silica gel, eluting with heptane to give 147 mg (90% yield) of a white
solid: mp 255e258 C, R
CDCl3):
2
100 kHz; sweep, 6.76 mT; scan time 60 s. The modulation ampli-
tude was set so that no line distortions occurred.
2.2.2. Measurements at defined oxygen contents
ꢀ
1
f
¼ 0.32 (heptane), H NMR (400 MHz,
For the experiments commercial 1 with about 50% 1H (Sigma
Aldrich, determined by HPLC analysis, see supporting information)
was used. The amount of 2H in compound 2 could not be deter-
mined due to missing pure 2H as reference substance. Samples in
toluene (1 ml, 1 mM) were flushed with pure nitrogen at a flow rate
of 0.8 l/min for 3 min using septum vials and needles. Samples in
octanol (1 ml, 1 mM) were flushed with either pure nitrogen or
defined mixtures of oxygen and nitrogen at a flow rate of 0.8 l/min
for 3 min. An anesthesia gas mixer with flow meter tubes (Dr a€ ger,
d
7.12 (d, J ¼ 7.8 Hz, 2H), 6.87 (t, J ¼ 7.8 Hz, 1H), 5.35 (s, 1H),
13
.22e2.04 (m, 18H), 1.80 (s, 12H), C NMR (126 MHz, CDCl
3
):
d
154.57, 135.92, 124.71, 119.92, 41.21, 36.94 36.87, 29.05, HRMS
-
(
ESI): calcd. for C26
H
32O [M ꢁ H] 361.254; found 361.252.
2
.1.4.3. 2,6-Bis(adamantan-1-yl)-4-{[3,5-bis(adamantan-1-yl)-4-
(17). Compound 16 (295 mg,
.814 mmol) was suspended in 3.5 ml formic acid. Para-
hydroxyphenyl]methyl}phenol
0
formaldehyde (24 mg, 0.814 mmol) was added. Mixture was stirred
and heated to reflux for 2.5 h. After cooling to RT 2 ml water were
added and the white precipitate was filtered off. The residue was
Lübeck, Germany) provided defined gas mixtures. The oxygen
content (in %) was measured in the solution directly after the EPR
measurements by a needle-type optical oxygen microsensor with
temperature control assuming ambient pressure (Type PSt1, Pre-
Sens GmbH, Regensburg, Germany). Oxygen sensitivities were
determined by plotting the simulated EPR linewidth as a function
of oxygen concentration and calculating the slope by linear
regression fit. For deuterated galvinoxyl the oxygen sensitivity was
also calculated by the ratio of the maximum amplitude and central
amplitude.
dissolved in CHCl
was neutral. The organic layer was then dried over MgSO
evaporated to dryness. The reaction mixture was purified by col-
umn chromatography on silica gel, eluting with heptane/CHCl (9/1
3
and washed with water until the aqueous layer
4
and was
3
to 0/10) to give 250 mg (83% yield) of a white to pale yellow solid:
ꢀ
1
mp ꢂ 330 C (degradation), R
f
¼ 0.06 (heptane), H NMR (500 MHz,
3
CDCl ): d 7.00 (s, 4H), 5.19 (s, 2H), 3.86 (s, 2H), 2.17e2.06 (m, 36H),

2740
L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
2
.2.3. Stability measurements
All samples were prepared under ambient air and were filled
into 50 l glass capillaries directly before measurement. Capillaries
ESI-MS analysis also showed there was residual educt with no H/D
exchange. Increasing the reaction time to 5 days markedly
improved the deuteration (tert. butyl groups 90% D, ortho position
53% D, methylene bridge 96% D), but dropped the yield to 4%. The
low deuteration efficiency in ortho position was most likely caused
by steric hindrance of the bulky tert. butyl groups. The low yield
was a result of degradation and polymerization processes.
Therefore we decided to start from an undeuterated precursor
with better accessible ortho positions, namely 2,6-di-tert-butyl-
phenol (6, Fig. 3).
m
were sealed by melting both sides. The area under the curve (AUC)
was calculated by double integration of the spectra. The AUC of the
first spectrum measured after preparation of each sample was set
as 100%. For ESI-MS measurements samples were stored under air
in 4 ml brown glass vials. ESI-MS measurements were recorded on
an LCQ classic (Thermo Finnigan, San Jose, California, USA).
2
.2.4. Spectral simulations with EasySpin
EPR spectra were analyzed and simulated with Matlab using
Use of 5% Pd/C (20 wt %) with 5% Pt/C (20 wt %) again led to the
formation of a cyclohexanone derivative (8, spectra see Supporting
Information). With 1% Pd/C (100 wt %)/1% Pt/C (100 wt %), the
desired product 9 was isolated with 28% yield after 2 days and 18%
after 3 days. The overall degree of deuteration was 92% with 69%
exchange in the ortho positions. Increasing the reaction time to 3
days did not increase the H/D exchange. Further attempts to
improve the exchange in the ortho positions were not successful.
Although indeed the degree of deuteration was markedly improved
by using compound 7 instead of compound 4, the ortho deuteration
was still insufficient for our purposes. Therefore we approached
compound 2 by still another pathway, starting with a deuterated
benzene nucleus (Fig. 4).
EasySpin [15] for the spectral simulations. In general function
Garlic” for fast and isotropic spectra with either Gaussian or Lor-
“
entzian peak-to-peak-linewidth was used for simulation of EPR
spectra. The g-factors were not determined and set to 2.0023 for all
simulations.
3
3
3
. Results and discussion
.1. Synthesis
.1.1. Synthesis of the perdeuterated galvinoxyl radical (2)
First, perdeuterated tert. butyl groups were introduced in the
ortho positions of compound 10 by a Friedel-Crafts alkylation with
tert. butanol-D10 under acidic conditions. To avoid alkylation in
para position, deuterated 4-bromophenol (10) was used as educt.
Bromine was removed from intermediate 11 by catalytic reduction.
The synthetic steps III and IV followed a literature procedure [17],
but using deuterated reactants and reagents. The deuteration de-
gree of compound 6 was found to be 96% (ESI-HRMS). The proton
NMR revealed residual protons were located at each position.
Compound 1, the fully deuterated galvinoxyl radical, contained a
marked amount of hydrogalvinoxyl 2H. The formation of hydro-
galvinoxyl during the last synthetic step is also known for undeu-
terated galvinoxyl radical [14]. Due to its very similar
chromatographic behavior, the removal of hydrogalvinoxyl is very
difficult and therefore 2 was used without further purification for
the EPR experiments.
We first approached the fully deuterated analog of the galvi-
noxyl radical by catalytic H/D exchange (Fig. 2) from undeuterated
0
4
,4 -methylenebis(2,6-di-tert-butylphenol) (4),
a
commercially
available, low-priced precursor of galvinoxyl. H/D-exchange in
alkyl-substituted aromatic compounds can be achieved by using a
mixture of palladium-on-carbon and platinum-on-carbon with D
as deuterium source at high temperature in the presence of a small
amount of H gas [16]. The mixture of Pd/C-Pt-C/D O-H has a
2
O
2
2
2
synergistic effect, wherein Pd/C catalyzes mainly the H/D-exchange
reaction at alkyl chains and Pt/C the exchange reaction at aromatic
rings.
The use of 5% Pd/C (20 wt %) with 5% Pt/C (20 wt %) unexpect-
edly led to the formation of the cyclohexanone derivative 5. Com-
13
pound 5 was identified by C NMR and IR spectroscopy (for spectra
see Supporting Information). The degree of deuteration for com-
pound 5 was not determined. When 1% Pd/C (100 wt %) with 1% Pt/
C (100 wt %) were used, the desired product 6 was isolated albeit
with incomplete H/D exchange. After 24 h, almost no H/D exchange
3.1.2. Synthesis of adamantyl-galvinoxyl (3)
13
had occurred. Proton and C NMR revealed that only protons at the
methylene bridge were exchanged with high yield. After 3 days, the
overall degree of deuteration was about 53% according to NMR
analysis. Only 5% H/D exchange occurred at the ortho position, the
tert. butyl groups were 56% deuterated, the methylene bridge 95%.
The tert. butyl groups of galvinoxyl have only been replaced by
phenyl and methyl groups, leading to radicals with complicated
EPR spectra due to hyperfine couplings [5,6]. We replaced the tert.
butyl groups by bulky adamantyl groups which do not lead to
Fig. 2. Deuteration of 4 by H/D exchange reaction. Reagents and Conditions: (I) 5% Pd/
Fig. 3. Deuteration of 7 by H/D exchange reaction. Reagents and conditions: (I) 5% Pd/C
ꢀ
ꢀ
C (20 wt %), 5% Pt/C (20 wt %), D
2
O, 180 C, H
2
(1 atm), 15e24 h. (II) 1% Pd/C (100 wt %),
(20 wt %), 5% Pt/C (20 wt %), D
2
O, 180 C, H
2
(1 atm), 15 h to 3 d. (II) 1% Pd/C (100 wt %),
ꢀ
ꢀ
1
% Pt/C (100 wt %), D
2
O, 180 C, H
2
(1 atm), 24 h to 5 d.
1% Pt/C (100 wt %), D
2
O, 180 C, H
2
(1 atm), 2 de3 d.

L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
2741
ꢀ
Fig. 4. Synthesis of compound 2 starting from deuterated educts. Reagents and conditions: (I) tert. butanol (D10, 98%), D
2
SO
4
(99.5% D), 160 C, 3 h, glass pressure vessel. (II) 5% Pd/C
ꢀ
(
50 wt %), D
2
(8 bar), DCM/methanol-D1 (2:1), rt, 7 h (III) paraformaldehyde (D2, 99%), isopropanol-D1, NaOD (40%) in D
2
O, 35 C, argon, 40 min. (IV) K
3
Fe(CN)
6
, NaOD (40%) in D
2
O,
benzene-D6, argon, rt, 60 min.
additional hyperfine couplings. While conferring about the same
degree of steric hindrance, the more rigid tertiary alkyl moiety of
adamantyl with methylene instead of methyl groups in the vicinity
of the spin system was expected to help with the analysis of the
hyperfine aspects of the EPR spectra.
paraformaldehyde led to the formation of a product mixture,
perhaps because of additional substitution at the meta positions
that are sterically more hindered next to tert. butyl groups.
Adamantyl-galvinoxyl 3 was generated from 17 by oxidation with
potassium ferricyanide. According to TLC and NMR analysis, 17 was
converted completely into radical 3. No residual hydro-adamantyl-
galvinoxyl 3H (Fig. 12(B)) was found.
The synthetic pathway is shown in Fig. 5. The most crucial part
of the synthesis is the alkylation of the aromatic ring, since due to
the steric hindrance twofold substitution with bulky alkyl groups is
difficult. To exclude formation of inseparable product mixtures, we
started the synthesis again from 4-bromophenol (12). The alkyl-
ation was done following a literature procedure [18]: compound 12
3.2. EPR spectra of the galvinoxyl derivatives
The EPR spectra of the different galvinoxyl derivatives
ꢀ
was heated to 210 C with a mixture of 1-adamantanol (13) and 1-
(
c ¼ 1 mM) were recorded in toluene and octanol under anoxic
bromoadamantane (14) to give 15 which was converted into
compound 16 by reductive debromation with Pd/C as catalyst. Re-
action of 16 with paraformaldehyde resulted in the formation of
compound 17 with high yield. The synthesis was done under
strongly acidic conditions by using formic acid as solvent. This is in
contrast to the synthesis of galvinoxyl where the condensation with
formaldehyde is done under basic conditions. With the adamantyl
conditions (Fig. 6 and Fig. 8).
derivatives,
base-catalyzed
condensation
of
16
with
Fig. 6. Experimental (black) and simulated (red) EPR spectra of (A) adamantyl-
galvinoxyl 3 and (B) galvinoxyl 1 (1 mM in toluene under nitrogen atmosphere at
room temperature). Spectral parameters were as follows: frequency, 1.3 GHz; micro-
wave power 0.42 mW; sweep time, 600 s; modulation frequency, 100 kHz; sweep,
2 mT; modulation amplitude ꢂ0.0045 mT (depending on the linewidth); number of
points, 1024.
Fig. 5. Synthesis of compound 3. Reagents and conditions: (I) 1-adamantanol, 1-
ꢀ
bromoadamantane, 210 C, 3 h, glass pressure vessel. (II) 5% Pd/C (50 wt %), H
2
(
(
5 bar), methanol/DCM, rt, overnight. (III) Paraformaldehyde, formic acid, reflux, 2 h.
IV) K Fe(CN) , NaOH (40%) in H O, benzene, argon, rt, 60 min.
3
6
2

2742
L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
Table 1
all contributing to the broadening of the EPR signal, while tert. butyl
groups contain only the equivalent of -protons. The increased
linewidth of adamantyl-galvinoxyl results from unresolved long-
range hyperfine couplings not only with -protons, but also with
- and ε-protons of the adamantyl groups. Long-range couplings are
based on -spin delocalization and are particularly effective if
the 2p -axis of the aromatic moiety and if the -bonds linking it
with the - and -protons lie almost in plane and are arranged in a
zigzag line [19]. For -protons this is called W arrangement [19]. In
Simulation parameter of 3 and 1.
g
a
1
[MHz]
a
2
[MHz]
lwpp (Lorentz) [mT]
g
3
1
17.35
17.30
4.20
4.10
90
40
d
p-s
z
s
g
d
3.2.1. Adamantyl-galvinoxyl (3)
d
The spectrum of commercial galvinoxyl radical 1 shows the
the adamantyl galvinoxyl 3 (Fig. 7), this zigzag arrangement is fixed
in the rigid annulated ring system, and rotation is only possible
around the C(a)-C(b)-bond.
common hyperfine coupling pattern of a doublet split into quintets
due to the hyperfine coupling with one methine and four aromatic
protons. The spectrum of the adamantyl derivative consists also of a
doublet (coupling with methine proton) with both peaks split to
quintets (coupling with aromatic protons). Though the ratio of in-
tensities within the quintet in both spectra is 1:4:6:4:1, the
appearance of the EPR spectra is different. The simulation of the
spectra shows that this is caused by an increase of the peak-to-peak
linewidth (lwpp) while the coupling constants a
methine proton) and a
the same (Table 1).
The hyperfine coupling of the alkyl protons was not resolved in
both radicals, but led to line broadening, which was accounted for
in the linewidth of the simulations. Adamantyl groups contain
3.2.2. Perdeuterated galvinoxyl (2)
The EPR spectrum of perdeuterated galvinoxyl radical 2 in
toluene (Fig. 8 (A)) shows partly resolved hyperfine structure with
four equivalent aromatic deuterium atoms and a single (methine)
deuterium atom. The EPR linewidth as obtained from spectral
simulations is reduced by 37.5% compared to the undeuterated
galvinoxyl radical. The reduced hyperfine coupling constants
correlate with the smaller gyromagnetic constant of deuterium
compared to hydrogen (Table 2). In octanol, the resolution of the
spectrum is increased due to reduced linewidths (Fig. 8 (B)). The
difference of the spectra measured in toluene and octanol arises
from different solvent properties such as polarity and from differ-
ences in specific interactions between the solvent and the radical.
1
(coupling with
2
(coupling with aromatic protons) are about
three magnetically different types of protons (Fig. 7, g-H, d-H, ε-H)
The signal pattern is composed of 18 lines with a linewidth of 11
which is very narrow.
mT
The precursor of the deuterated galvinoxyl (compound 6) con-
tained residual protons at each position, the overall degree of
deuteration being 96%. The additional signals of not fully deuter-
ated species are visible in the low- and high-field of the spectrum
measured in octanol.
3.3. Oxygen sensitivity of undeuterated and perdeuterated
galvinoxyl radical
Galvinoxyl type radicals react with oxygen to form different
degradation products. In the presence of the reduced species (1H
and 2H), galvinoxyl radicals are stabilized with the potential to
become probes for oxygen determination. Therefore we thoroughly
investigated the influence of oxygen on the EPR spectra of galvi-
noxyl and analogs. Since galvinoxyl type radicals have a high lip-
ophilicity and are not soluble in aq. solutions, it was first necessary
to define a solvent which on the one hand dissolves the radicals, but
on the other hand is biocompatible for potential in vivo applica-
tions. Stability tests in 1-octanol showed no degradation during the
course of several hours. Octanol is known to be well tolerated
in vivo. To prove the applicability of galvinoxyl radicals as oxygen-
sensitive spin probes, EPR spectra of commercial and deuterated
galvinoxyl (1 mM in octanol) were measured at different oxygen
levels. For in-vivo applications, oxygen levels between 0 and 10%
are of particular interest.
Fig. 7. 3D structure of alkyl-substituents of (A) adamantyl-galvinoxyl 3 and (B) gal-
vinoxyl radical 1.
3.3.1. Galvinoxyl 1
The peak-to-peak linewidth of the spectra was extracted from
the experimental data by simulating the experimental results with
Fig. 8. Experimental (black) and simulated (red) EPR spectra of perdeuterated galvi-
noxyl in (A) toluene and (B) octanol (1 mM under nitrogen atmosphere at room
temperature). Spectral parameters were as follows: frequency, 1.3 GHz; microwave
power 0.42 mW; sweep time, 600e1200 s; modulation frequency, 100 kHz; sweep
Table 2
Simulation parameters of perdeuterated galvinoxyl in toluene and octanol.
a
1
[MHz]
a
2
[MHz]
lwpp (Lorentz) [mT]
0
.75 mT; modulation amplitude, ꢂ 0.0011 mT (depending on linewidth); number of
Toluene
Octanol
2.59
2.59
0.64
0.64
25
11
points, 1024.

L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
2743
Matlab using the EasySpin function “Garlic”. For simulation pa-
rameters see Materials and Methods and the Supplementary In-
is advantageous for the measurement of small changes in
oxygen concentrations.
formation. To achieve the best simulation, 0% O
2
-spectra were
(2) The change of the line shape is directly connected with the
change of the linewidth: the broadening leads to a super-
stition of signals and thus to a change to the amplitudes. Due
to that the signal amplitudes can reflect the spin exchange
phenomena between the galvinoxyl radical and oxygen. This
change of line shape was expressed as the ratio of the
maximum amplitude and the amplitude in the center of the
spectrum (Fig. 10 (C) and asterisks in Fig. 10 (A)). This
calculation provides a reproducible non-linear relationship
between line shape and oxygen concentration up to 6% ox-
ygen. The loss of reproducibility above 6% oxygen is associ-
ated with the loss of hyperfine structure at this oxygen level.
From 1% to 6% oxygen, linearization is possible by taking the
logarithm of y-axis (see Supporting Information). This facil-
itates data analysis.
simulated using Gaussian line shape. Above 0% oxygen, Lorentzian
line shape was used for the simulation. Undeuterated galvinoxyl
shows a linear relationship between EPR linewidth (peak-to-peak)
and oxygen concentration. The linewidth range (Fig. 9) is compa-
rable with the linewidth of lipophilic chlorinated trityls dissolved in
oils (e.g. isopropyl myristate) [20]. The oxygen sensitivity is about
2
.8
philic oxygen spin probes (e.g. hydrophilic tertrathia-trityls, 0.5
[20]) is a result of the high oxygen solubility in octanol
m
T/% O
2
. The stronger response to oxygen compared to hydro-
m
T/
%
O
2
compared to water (1-octanol 1.5 mmol/l, water 0.29 mmol/l oxy-
ꢀ
gen at 20 C, 0.213 bar O
2
[21]):at the same oxygen partial pressure
octanol dissolves more oxygen and thus a more intense Heisenberg
spin exchange and line broadening occurs. In contrast to trityl
radicals, galvinoxyl is a commercially available and low-priced spin
probe. Due to this and the comparable oxygen sensitivity of chlo-
rinated trityl radicals, galvinoxyl is a reasonable alternative for EPR
oximetry applications in lipophilic formulations, esp. at oxygen
levels of physiological interest.
To our knowledge, the calculation of oxygen levels by analyzing
the maximum amplitude and the amplitude in the center of the
spectrum of a perdeuterated radical has not been reported before. It
takes advantage of the narrow and very distinct coupling pattern
due to deuterium atoms. So again, deuterated galvinoxyl has a
better oxygen sensitivity than fully hydrogenated galvinoxyl. This is
advantageous when small changes in oxygen concentrations have
to be monitored.
3.3.2. Perdeuterated galvinoxyl 2
The EPR spectrum of deuterated galvinoxyl in octanol shows a
well resolved hyperfine structure under anoxic conditions. With
increasing oxygen concentration (Fig. 10 (A)), the signal is broad-
ened which leads to a complete loss of the hyperfine structure
between 5% and 6% oxygen and one broad signal above 6%. The
change of oxygen concentration causes both a change of (1) line-
width and (2) line shape.
3
.4. Stability studies with commercial and adamantyl-galvinoxyl
The stability of galvinoxyl radicals depends on different factors.
It is known that degradation due to reaction with oxygen is
inhibited by hydrogalvinoxyl [14], but to our knowledge this
behavior has not been further examined.
(
1) The linewidth of the spectra was determined by simulating
the experimental results with Matlab using EasySpin. For
simulation parameters, see Materials and Methods and
Supplementary Information. The linewidth was calculated as
peak-to-peak linewidth on the basis of a Lorentzian model.
Fig. 10 (B) shows the linear relationship between linewidth
and oxygen concentration. Due to the narrow lines at 0%
oxygen, deuterated galvinoxyl exhibits better oxygen sensi-
3.4.1. Formation of the hydro-analog of galvinoxyl type radicals
We found commercial galvinoxyl (Sigma Aldrich) to contain
about 50% hydrogalvinoxyl 1H (HPLC analysis, see supporting in-
formation). For the synthesis of perdeuterated galvinoxyl we found
that the hydro-analog is formed during the last synthetic step as a
result of incomplete oxidation of the precursor 6. Formation of
hydrogalvinoxyl does not occur during storage of the compound.
tivity (4.8 mT/% oxygen) than the undeuterated radical which
Fig. 9. (A) Oxygen calibration curve of commercial galvinoxyl dissolved in octanol (c ¼ 1 mM) (n ¼ 3). (B) EPR spectra of galvinoxyl at different oxygen concentrations. Spectral
parameters were as follows: frequency, 1.3 GHz; microwave power 0.42 mW; sweep time, 1200 s; modulation frequency, 100 kHz; sweep, 2 mT; modulation amplitude ꢂ0.0045 mT
(
depending on the linewidth); number of points, 1024.

2744
L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
Fig. 10. (A) EPR spectra of perdeuterated galvinoxyl 2 (1 mM in octanol) at different oxygen concentrations; parameters see Fig. 8 (B) Oxygen calibration curve of 2 dissolved in
octanol (c ¼ 1 mM, n ¼ 3) determined by linewidth change. (C) Oxygen calibration curve of perdeuterated galvinoxyl dissolved in octanol (c ¼ 1 mM, n ¼ 3) determined by change of
amplitudes.
This is in accordance with published literature [5]. Increase of the
reaction time or of the amount of potassium ferricyanide did not
lead to complete formation of 6 to radical 2. Fig. 11 shows possible
reaction pathways for the formation of 1 resp. 1H. They are both
formed in a multi-step oxidation process starting from the meth-
ylenebisphenol 4, and accompanied by rearrangement and
disproportionation of intermediates.
3.4.2. Influence of the hydro-analog on the radical stability
The presence of 1H next to 1 leads to an inhibition period during
which only minor degradation occurs. After this period, the reac-
tion with oxygen causes rapid degradation of the radical. Identifi-
able
hydroxybenzaldehyde and 2,6-di-tert-butyl-1,4-benzoquinone
14,22]. The length of the inhibition period is dependent on the
degradation
products
are
2,6-di-tert-butyl-4-
[
hydrogalvinoxyl concentration [14]. We also found this behavior for
adamantyl-galvinoxyl 3 in the presence of different amounts of 3H
(Fig. 12 (A)). The 3, 3H-mixtures were generated by mixing the
radical with different amounts of the methylenebisphenol precur-
sor 17 (Fig. 12 (B)). Compound 3H was formed directly after mixing
of both compounds. The samples for the stability tests were pre-
pared under atmospheric air and were measured in sealed 50 ml
capillaries to prevent gas exchange with ambient air during the
measurements.
EPR measurements over different time periods showed intense
degradation of the pure radical after 1 h. After 1.8 h, 50% of the EPR
Fig. 11. Possible reaction pathway for the formation of galvinoxyl (1) starting from
methylenebisphenol (4). Figure adapted from Omelka et al. [5].

L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
2745
Fig. 12. (A) Stabilization of 3 (cstart ¼ 1 mM in toluene) by 3H. AUC was calculated by double integration of the EPR spectra. (B) Formation of the hydro-analog (3H) of adamantyl-
galvinoxyl (2). (C) EPR spectra of 3 with 0% 3H and 50% 3H.
signal of 3 had vanished. With increasing concentration of the
hydro-analog 3H the inhibition period until fast degradation was
prolonged. The rate of the fast degradation did not change up to
about 21% 3H. Above 21% of 3H, the degradation rate was reduced.
Fig.12 (C) shows the EPR spectra of adamantyl-galvinoxyl 3 with
and without the presence of 3H. The EPR signal intensity of pure 3
decreased over time while resolution of the hyperfine structure
increased. At about 30% decrease of the AUC after 1.75 h, the hy-
perfine structure was well resolved due to linewidth reduction. This
was easily explained by oxygen consumption during the degrada-
tion process. This was also observed for the other samples, exem-
plarily shown for 50% 3H content.
ꢃ The hydro-analog reactivates galvinoxyl-oxygen adducts like
hydro- or endoperoxides by reduction and is oxidized itself to
the radical by reaction with these adducts, leading to the
observed degradation profile over time. This mechanism is
further supported by the behavior of 1 in different solvents (see
below).
3.4.3. Stabilization of galvinoxyl radicals by other compounds
To shed more light on the degradation and stabilization mech-
anism, the stabilization of 3 with other substances was investi-
gated. The addition of p-chloranil and benzophenone as bulky
aromatic compounds did not result in any stabilization of the
radical, supporting the third possibility (v.s. 3.4.2). The addition of
phenol and 2,6-di-tert-butylphenol did lead to stabilization, but
TLC analysis showed that 3H was formed. The enhanced stability is
therefore again a result of the presence of 3H.
However, in contrast to the behavior of pure 3, the signal
amplitude slightly increased when 50 or 60 wt% of 3H were present.
(
Below 50% 3H, this behavior was less distinct.) If this resulted from
the fact that reduction of the linewidth compensated the decrease
of signal intensity, the signal amplitudes would increase or stay
constant. The smaller linewidths suggest that at the same signal
intensity (measured as AUC) the oxygen concentration was lower in
the sample with the higher amount of 3H. This observation on
galvinoxyl stabilization by their hydro-analogs suggests the
following mechanistic possibilities:
3.4.4. Influence of the solvent on radical stability
EPR measurements of 1 (stabilized with 1H) in different solvents
showed that the stability of galvinoxyl radicals was also solvent
dependent (Fig. 13).
On the basis of the results shown in Fig. 13 the following con-
clusions were drawn:
ꢃ The hydro-analog may protect the radical by steric shielding and
diminish the interaction between radical and oxygen. However,
the linewidth of the first spectrum of each sample does not
change with increasing concentration of the hydro-analog
which should be the case if 3H indeed diminishes the direct
interaction between radical and oxygen.
ꢃ The influence of oxygen solubility in the solvents on stability
was negligible. Chloroform and cyclohexane both dissolve
ꢀ
2.4 mM oxygen (0.213 bar oxygen, 20 C) [21], but their ability to
stabilize 1 is completely different. The same applies to octanol
and butanol. They dissolve oxygen at 1.5 mM and 1.8 mM
ꢀ
It seems more likely that a reaction between hydrogalvinoxyl
and oxygen or between hydrogalvinoxyl and galvinoxyl occurred:
(0.213 bar oxygen, 25 C) [21].
ꢃ With decreasing polarity resp. increasing lipophilicity, the sta-
bility increases. 1 is stable over 6 h in nonpolar and less polar
solvents like heptane, cyclohexane, toluene and octanol, but
undergoes degradation in more polar solvents like butanol,
chloroform, and dichloromethane.
ꢃ The hydro-analog may react directly with oxygen to form either
the radical and-or other degradation products. However, ESI-MS
analysis of a pure 1H solution (c ¼ 1 mM, toluene) did not show
any degradation products resulting from reaction with oxygen.
The solution showed a very weak EPR signal under standard
conditions for galvinoxyl. Its intensity (measured as AUC) did
not increase after bubbling pure oxygen through the solution
Galvinoxyl solutions in toluene, heptane, octanol, butanol,
benzyl alcohol and dichloromethane were analyzed by ESI-MS to
see which degradation products were formed. After one day,
toluene, butanol, benzyl alcohol and DCM solutions contained no
residual 1. Heptane and octanol still contained 1 which was
corroborated by EPR spectrometry. After 3 days in heptane, 1 was
completely degraded while the octanol solution still showed an
(
10 min, 0.1 l/min). (For the synthesis of 1H see Supplementary
Information.)
A third possibility seems to be supported best by the data:

2746
L. Lampp et al. / Tetrahedron 75 (2019) 2737e2747
Fig. 13. Stability of 1 in different solvents (cstart ¼ 1 mM). AUC was calculated by double integration of the EPR spectra.
intense EPR signal. Even after 5 days in octanol, an EPR signal was
measurable. Analysis of the degradation products revealed a dif-
ference between protic and aprotic solvents: In aprotic solvents, the
synthesized with a high degree of deuteration. The EPR spectrum
yields a many-line-pattern due to partly resolved hyperfine cou-
plings with deuterium atoms and very small linewidth under
anoxic conditions. The oxygen sensitivity of undeuterated and
perdeuterated galvinoxyl was examined. Both radicals show good
oxygen sensitivity within the physiological range which makes
them to potential probes for EPR oximetry. The mechanism of
stabilization by hydro-analogs of galvinoxyl-type radicals was
examined. Due to the results shown in this study the most
reasonable mechanism seems to be a direct reaction of the hydro-
analog and radical-oxygen-adducts with can either lead to the
reactivation of the radical or to the oxidation of the hydro-analog to
the radical.
degradation
products
were
2,6-di-tert-butyl-4-
hydroxybenzaldehyde, 2,6-di-tert-butyl-1,4-benzoquinone and
different oxygen adducts like the endo- or hydroperoxide. In protic
solvents, the main degradation product was the hydro-analog 1H.
This was also the main degradation product in octanol after com-
plete loss of the EPR signal. Thus the reaction rate with oxygen
seems to be diminished in solvents with increasing lipophilicity,
and it was insignificant in protic solvents.
The following mechanistic conclusions rationalize this behavior.
In lipophilic solvents, the interaction between galvinoxyl radicals
and hydro-analog is facilitated as both are not solvated well. They
form complexes through polar interactions (e.g. H-bonds, charge
transfer), preparing them for mutual interactions (e.g. H-atom and
electron transfer). This increases the probability that the radical is
formed again by oxidation of hydrogalvinoxyl through H-atom
transfer. This actually supports the third theory regarding the sta-
bilization of 3 by 3H, presented in Section 3.4.2. The dependence of
the ease of H-atom transfer between phenols and galvinoxyl on the
polarity of aprotic solvents was found in earlier studies [23].
Obviously, H-atom transfer is much faster in protic solvents than
reaction with oxygen.
For the future use of galvinoxyl and analogous radicals the above
observations and mechanistic explanations translate into the
following practical conclusion. For studies in aqueous solutions
including body fluids, galvinoxyls should be packed in a relatively
lipophilic medium with neutral pH, adding 50 wt% or more of the
hydro-analog. Thus, they can serve as oxygen sensors over a couple
of days. Oxygen levels below 6% - that are actually of greater in-
terest physiologically than higher oxygen levels - can be assessed
best when determining both line width and line shape as reported
above.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.tet.2019.03.051.
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[
[
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4
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